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Turner, R.D., Wingham, J.R. orcid.org/0000-0002-8331-9206, Paterson, T.E. orcid.org/0000-0002-2951-115X et al. (2 more authors) (2020) Use of silver-based additives for the development of antibacterial functionality in Laser Sintered polyamide 12 parts. Scientific Reports, 10. 892. ISSN 2045-2322

https://doi.org/10.1038/s41598-020-57686-4

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Use of silver-based additives for the development of antibacterial functionality in Laser Sintered polyamide のは partsRobert D. Turnerの┸は┸ば┸ James R┻ Winghamの┸ば┸ Thomas E┻ Patersonは┸ Joanna Shepherdはこ & Candice Majewskiのこ

Infectious diseases ゅexacerbated by antimicrobial resistanceょ cause death┸ loss of quality of life and economic burden globally┻ Materials with inherent antimicrobial properties oプer the potential to reduce the spread of infection through transfer via surfaces or solutions┸ or to directly reduce microbial numbers in a host if used as implants┻ Additive Manufacturing ゅAMょ techniques oプer shorter supply chains┸ faster delivery┸ mass customisation and reduced unit costs┸ as well as highly complicated part geometries which are potentially harder to clean and sterilise┻ Here┸ we present a new approach to introducing antibacterial properties into AM┸ using Laser Sintering┸ by combining antimicrobial and base polymer powders prior to processing. We demonstrate that the mechanical properties of the

resultant composite parts are similar to standard polymer parts and reveal the mode of the antibacterial

activity. We show that antibacterial activity is modulated by the presence of obstructing compounds

in diプerent experimental media┸ which will inform appropriate use cases┻ We show that the material is not toxic to mammalian cells┻ This material could be quickly used for commercial products┸ and our approach could be adopted more generally to add new functionality to Laser Sintered parts.

he global Additive Manufacturing (AM) market has grown by an average of 26.9% annually for the last 30 years, with the overall revenue of the industry currently estimated at $9.8 billion, and aerospace, automotive and healthcare being major sectors1. Parts are produced in a layer-by-layer manner, directly from a Computer-Aided Design (CAD) ile. his layer-by-layer approach provides key beneits through removing the need for tooling and increasing the ease with which complex geometries can be produced. However, despite their clear potential, the range of materials that can be used in AM processes is limited compared to more traditional manufacturing techniques, which in turn has restricted the range of applications in which they can be used.

Laser Sintering is an AM technology that produces parts by selectively scanning and melting consecutive cross-sections of polymer powder particles. Areas which have not been scanned remain as loose powder through-out the process, acting to support any overhanging areas, which in turn allows the economic production of highly complex part geometries. his geometric capability makes Laser Sintering highly suited to production of com-plex, optimised, products and devices, or to the production of products and devices personalised to individuals. However, particularly when considering hand-held and/or medical products, increasing geometric complexity can render them diicult and time-consuming to clean efectively, potentially providing increased chance of spread of bacteria. Incorporation of antibacterial properties into the parts themselves could reduce or eliminate this risk, and is the focus of this work.

Many antimicrobial products are currently available to purchase, with a growing global market for antimi-crobial additives (materials that are added to base materials to yield antimicrobial properties). Antimicrobial products are used in the healthcare sector as well as in consumer goods - the demand for these is driven not only by a desire to improve health, but to develop opportunities to create added value. hey have the potential to make a positive impact in healthcare in implants, prosthetics and splints2, and surfaces and devices in clinical

のDepartment of Mechanical Engineering┸ University of Sheffield┸ Royal Exchange Building┸ びぱ Garden Street┸ Sheポeld┸ SのぱBJ┸ UK┻ はSchool of Clinical Dentistry┸ University of Sheポeld┸ のぶ Claremont Crescent┸ Sheポeld┸ SのねはTA┸ UK┻ ばThese authors contributed equally┺ Robert D┻ Turner and James R┻ Wingham┻ こemail┺ j┻shepherdｌsheポeld┻ac.uk; c┻majewskiｌsheポeld┻ac┻uk

OPEN

https://doi.org/10.1038/s41598-020-57686-4mailto:[email protected]:[email protected]:[email protected]


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settings (e.g. sinks, instruments, keyboards). Adlhart and colleagues propose a helpful set of categories for anti-microbial surfaces: anti-adhesive surfaces (based on preventing microbial attachment3), contact-active surfaces (that kill microbes on contact) or biocide releasing surfaces (from which the active ingredient is eluted into the surroundings)4. If a material is not inherently antimicrobial, the diferent types are obtained either by mixing antimicrobial additives into the bulk material, or through direct physical modiication (e.g. altered roughness) or chemical modiication (e.g. attachment of a biocide) to the surface. he biocidal properties of copper, zinc and silver (amongst other metals) are exploited in commercial antimicrobial additives and in ongoing research5, with silver a well-established choice. We therefore chose to develop a composite material to be used in AM using silver-based additives.

Research into AM of antimicrobial materials is a small but rapidly growing ield. Researchers have incor-porated antimicrobials (or antibiotics) into polymer, metal and ceramic composites using a range of AM tech-niques. Fused Deposition Modelling (FDM) has been used to incorporate, for example, nitrofurantoin6 or silver nitrate7 for potential use in medicine and healthcare. Stereolithography (SLA) has been used to incorporate 4-aminosalicylic acid8 for customised drug-release kinetics, silver nanoparticles9 or quaternary ammonium compounds10. Binder Jetting has been employed, oten exploiting the capacity to dissolve antibiotics in the liq-uid binder, to form polymer11 or ceramic12 composites, for potential bone implants amongst other applications. Robocasting has been used to incorporate a range of agents including levoloxacin13, quaternary ammonium compounds14 or silver nanoparticles15, in a ield again largely aimed at developing bone implants. Selective Laser Melting (SLM) of titanium16 or cobalt-chromium-molybdenum17 has been employed by several groups to develop bone implants – adding antimicrobial functionality to these generally requires a post-processing step, likely due to the high temperatures involved in melting metal being deleterious to many antimicrobials. To the best of our knowledge, no antimicrobial materials made using Laser Sintering have been described to date in the academic literature.

he proposed antimicrobial efects of silver remain an active research topic and fall into two broad categories: direct reactions between silver ions and cellular components (proteins, membranes, DNA) or indirect damage caused by reactive oxygen species (e.g. hydrogen peroxide) generated by the silver18,19. Xiu and coworkers showed that silver ions are equally toxic to Escherichia coli in aerobic and anaerobic conditions, contending on the basis of this that reactive oxygen has no role in the mechanism of action. However, an earlier study of E. coli and Staphylococcus aureus20 contradicts this inding, arguing that there is a role for oxidative stress under aerobic conditions. It may be that this inconsistency can explained by methodological diferences. An important study highlights the protective efect of thiol groups in the experimental medium21. It seems reasonable on the basis of this to assume that silver can bind these thiols and as a result become unavailable to participate in any other reactions that might (directly or indirectly) harm microbes. It has also been established that increased levels of serum in growth medium for cultured human cells is protective against the toxic efects of silver22, perhaps by a similar thiol-binding mechanism. Whatever the mechanism of antimicrobial action, our concern as engineers is to develop an approach that can safely kill or inhibit microbes using silver ions, and all relevant previous research suggests that to do this silver ions must be available in proximity to the microbes. In this manuscript, we do not contribute to the debate on the mechanism of action of silver as an antimicrobial, but explain how our material delivers silver ions and under what circumstances it will be most useful.

Here, we have adopted a commercially available antimicrobial additive (Biocote B65003) and combined this with a widely used Laser Sintering powder (polyamide 12, EOS PA2200) to create an antimicrobial material suit-able for a range of potential uses. We characterise the resulting composite parts, including their mechanical prop-erties, antibacterial properties (using S. aureus as a model Gram positive organism and Pseudomonas aeruginosa as a model Gram negative), and demonstrate a lack of cytotoxic efects.

Results and DiscussionProcessability of material. We printed polyamide 12 parts in a range of geometries using standard settings for this material on a commercial EOS Formiga P100 printer. We subsequently printed the same part geome-tries in polyamide 12 mixed with 1% B65003 silver phosphate glass. he parts were qualitatively near-identical (Fig. 1a). his straightforward experiment demonstrated the processability of a mix of sinterable and non-sinter-able powders via a Laser Sintering 3D printer.

Mechanical properties of parts. Tensile testing was employed to ensure that there were no detrimental efects on the mechanical properties of the composite when compared with the standard polyamide 12 Laser Sintering material. We therefore printed “dogbones” for use in tensile testing, which involved measuring the force required to break the parts when subjected to a tensile load. his yielded measurements of Young’s Modulus (E – the stifness of the material), Ultimate Tensile Strength (σuts – how much force per unit area is needed to break the material) and Elongation at Break (εmax – how much the material ‘stretches’ before breaking) which were similar for both materials (Fig. 1b,c): he properties of the Laser Sintered parts were measured as; polyam-ide 12 (E = 1590 ± 69 MPa, σuts = 47.3 ± 0.4 MPa, εmax = 31.0 ± 1.2%) and 1.0% B65003 (E = 1580 ± 136 MPa, σuts = 47.5 ± 0.4 MPa, εmax = 29.8 ± 3:1%). A 2 sample Welch’s t-test was carried out to compare the materials to each other, with p values < 0.05 considered to be signiicantly diferent. he resulting p values were; 0.89 for E, 0.39 for σuts, and 0.43 for εmax showing that there was no signiicant diference. hese results indicate the materials can be used interchangeably, with no efect on the mechanical properties of printed parts.

Part structure and composition. Parts made of the base material and composite were imaged using SEM. his revealed the characteristic granular structure of the surface of the Laser Sintered polyamide (Fig. 2). Some diferences in contrast were observed between the surface of the base material and the composite (compare Fig. 2a,b with Fig. 2c,d) due to the sensitivity to material of backscattered electrons.

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he silver phosphate glass was found to be distributed evenly throughout the parts on this basis of X-Ray micro tomography. he diference in electron density between this and the base polyamide 12 led to the silver phosphate glass appearing brighter, allowing analysis of its dispersion (Fig. 2e,f).

EDX elemental analysis was used to identify the distribution of the additive on the surface. A spectral analysis of both the B65003 additive and the composite part (Table 1), showed that although silver was present in the additive, the low concentration was such that it was below the detection limit when combined in the printed part. To map the location of the additive (Fig. 3), the elements phosphorus and oxygen were instead used as these were abundant in the additive but not in PA2200, as shown in Fig. 3c,d.

Composite parts release silver. Having validated the engineering properties of the parts, we next deter-mined the extent to which the active antimicrobial, silver, was eluted. his was measured using Inductively Coupled Plasma-Optical Emission Spectroscopy (ICP-OES) which detects the concentration of silver ions in solution. In a preliminary test, silver phosphate glass was found to release far more silver ions than metallic silver powder, further validating this material as an antimicrobial additive (Table 2). We estimate our parts (containing 1.0% B65003) released a similar amount of silver per unit surface area over 24 hours (~0.012 mg/l/cm2) as a pre-viously reported compression moulded polyamide 12 composite containing 1.4% Nanosilver (~0.03 mg/l/cm2)23 released over 100 days. his indicates that our new AM approach yields a material that releases silver (loosely speaking) at least as well as existing (non-AM) materials. he surface area of our spheres (used in this calculation) was 4.62 ± 0.01 cm2 (standard error, n = 100 {20 parts, 5 measurements per part, pooled}).

Composite parts have an antibacterial effect against gram positive Staphylococcus aureus and gram negative Pseudomonas aeruginosa. Assured that our composite parts release silver, we then checked for antibacterial activity against two representative pathogens. Parts were incubated in Phosphate Bufered Saline (PBS) containing bacteria for 24 hours, ater which the amount of bacteria in the medium (plank-tonic bacteria) and on the parts surface (bioilm) was measured. Bacteria survived for 24 hours in PBS with a normal polyamide 12 part. here were fewer planktonic S. aureus in PBS that had held a part made of the antibac-terial composite than in the equivalent for polyamide 12, with some samples containing so few bacteria that they

Figure 1. Engineering properties of parts (a) Photograph of a selection of parts made from PA2200 (let) alongside the 1% B65003 composite material (right). (b) Raw stress-strain curves from tensile testing. (c) A comparison of Young’s modulus (E), ultimate tensile strength (σuts) and elongation at break (εmax) for both materials.

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were undetectable in our assay (Fig. 4a). his efect was mirrored for S. aureus bioilms (Fig. 4b). In the case of P. aeruginosa there was also a reduction in numbers of bacteria comparing antibacterial polyamide 12 (containing 1.0% B65003) to polyamide 12 for both planktonic organisms and those in bioilms on the surface of the parts (Fig. 4c,d).

Antibacterial eプects do not require contact between parts and bacteria┻ To explore whether the anti-bioilm properties of our material were mediated by contact between bacteria and parts, we incubated parts

Figure 2. Images of parts – (a,b) SEM images of base material (sintered PA2200), (c,d) composite material. he lighter, more angular objects in d are likely silver phosphate glass. (e) X-Ray micro tomography section of base material (PA2200), showing pores. (f) X-Ray micro tomography section of the composite showing even distribution of silver phosphate glass particles.

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in PBS, removed them ater 24 hours and then inoculated the PBS with bacteria. here were less bacteria in PBS that had contained antibacterial polyamide 12 (containing 1.0% B65003) than that which had held normal poly-amide 12 (Fig. 4e). his proves that contact between parts and bacteria is not required for an antibacterial efect (it does not totally disprove that there may also be some contact mediated efects). We infer from this that silver ions are eluted from the antibacterial polyamide 12 (containing 1.0% B65003) in solution and difuse to their targets. he anti-bioilm efect is therefore likely a consequence of bacterial mortality (likely prior to adhesion to the antibacterial polyamide 12 (containing 1.0% B65003)) rather than an anti-adhesion mechanism.

Sample

Weight percentage ± Standard Deviation (%)

O C P Ti Mg Ca Ag

B65003 63.6 ± 0.4 16.5 ± 0.4 12.6 ± 0.1 0.0 ± 0.0 3.5 ± 0.0 2.9 ± 0.0 0.9 ± 0.1

PA2200 + 1.0% B65003 72.5 ± 0.1 27.1 ± 0.1 0.2 ± 0.0 0.1 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0

Table 1. Composition of the silver phosphate glass additive (B65003) and the composite part (PA2200 + 1.0% B65003) obtained from EDX analysis.

Figure 3. SEM and EDX map of composite PA2200 + B65003 surface. (a) SEM micrograph of the surface scanned with EDX. (b–d) Elemental map from EDX analysis of the surface with colour indicating detection of the corresponding element.

Sample Mean Standard Errorn samples above 0.002 mg/l detection limit (/total)

Silver ion concentration ater 24 hours incubation of silver or silver phosphate glass at 2 mg/ml in water

Control (Water) NA 0/4

Silver Powder 0.006 ± 0.0016 mg/l 3/4

B65003 26 ± 2.5 mg/l 4/4

Silver ion concentration ater 24 hours incubation of polyamide 12 or antibacterial polyamide 12 (containing 1.0% B65003) spheres in 5 ml water

PA2200 NA 0/3

PA2200 + 1.0% B65003 0.056 ± 0.018 mg/l 3/3

Table 2. ICP-OES measurements of silver concentration.

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Antibacterial effects are diminished in rich media. PBS is reasonably representative of a very nutrient-poor (but osmotically stable and close to pH neutral) environment for bacteria, and is most relevant to partly hydrated fomite surfaces such as one might ind in a kitchen or bathroom environment, or in some clinical or semi-clinical settings. However, we were also interested in the eicacy of our parts in a rich-media

Figure 4. Comparison of amount of S. aureus (CFU/ml) in PBS surrounding (a), or attached to (b) polyamide 12 or antibacterial polyamide 12 (containing 1.0% B65003); and comparison of amount of P. aeruginosa (CFU/ml) in PBS surrounding (c), or attached to (d) polyamide 12 or antibacterial polyamide 12 (containing 1.0% B65003). Comparison of amount of S. aureus (CFU/ml) in PBS previously incubated with polyamide 12 or antibacterial polyamide 12 (containing 1.0% B65003) (e).

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environment; one containing a complex mix of biologically derived molecules in which bacteria had the nutri-ents needed to grow and divide. To investigate this we used Brain Heart Infusion (BHI), a commonly used rich medium for growing either S. aureus or P. aeruginosa. In this medium there was no diference between bacteria grown in media containing either polyamide 12 or antibacterial polyamide 12 (containing 1.0% B65003) for either species (Fig. 5a–d).

We hypothesised, informed by previous work21,22, that the diference in performance between PBS and BHI was due to the presence of chemicals in BHI that react with silver ions, rendering them unable to perform their antibacterial function, particularly molecules with thiol (sulhydryl) functional groups. To test this, we repeated our experiments in PBS containing 1 mM reduced glutathione (Fig. 5e,f). We saw no substantial diference in the eicacy of parts made of polyamide 12 or antibacterial polyamide 12 (containing 1.0% B65003) in this medium. his result, in the context of previous indings, strongly suggests that the presence of confounding chemicals explains the relative lack of eicacy in BHI as compared with PBS.

Composite parts do not have a cytotoxic eプect┻ For any use case where parts are to come into contact with humans or animals, it is important to determine levels of toxicity. his was achieved by incubating parts with 2D monolayers of human ibroblast cells at the bottom of tissue culture wells. Such cells make up connective tis-sue, including dermal skin layers, and produce collagen. We found no diference in metabolic activity (measured using a Presto Blue assay) between any of the wells containing either no part, a part made from polyamide or antibacterial polyamide 12 (containing 1.0% B65003) (Fig. 6).

ConclusionsWe have developed an approach for producing Additive Manufactured parts with antibacterial properties for use in the Laser Sintering process. he engineering properties of the new composite are indistinguishable from those of the standard polyamide 12 base material. he material is most efective in nutrient-poor hydrated environ-ments (where reactions that interfere with the activity of the silver are less likely) and under these circumstances is able to reduce numbers of planktonic bacteria in its surroundings and numbers of bioilm bacteria attached to the surface. We have shown that there are no fundamental issues with cytotoxicity.

Eicacy against such diverse bacterial species as S. aureus and P. aeruginosa, in the context of the long history of silver as an antimicrobial agent, strongly suggests a spectrum of action against both Gram positive and Gram negative bacteria. Additional work would determine the breadth of antimicrobial activity of our material across other species, but given our evidence we would expect our material to be efective against further unwanted bacteria.

here are a range of potential uses for the material where favourable circumstances are present, including those that are intermittently hydrated, in kitchens, bathrooms and on hospital wards. However, a demonstrable lack of eicacy in rich BHI media and in PBS containing reduced glutathione should temper expectations of per-formance in nutrient-rich environments (e.g. in vivo or in the food industry). Future work will include ield trails to validate laboratory results in real-world settings.

MethodsMaterials. A polyamide 12 based powder was chosen as the base material (PA2200, EOS) for the antimicro-bial composite. his was chosen as it is widely used in industry, meaning any added functionality could have a high impact in a relatively short timescale. A commercially available silver phosphate glass (B65003, BioCote) was chosen to create the antimicrobial efect. his material was provided in powder form, with particle size



Inductively coupled plasma – optical emission spectroscopy. Powders or parts were incubated for 24 hours at 37 °C with agitation in distilled water. Supernatant was extracted and, for powder samples, was centri-fuged (15 minutes at 2000 rpm) and iltered (0.2 µm ilter) to remove suspended powder particles. ICP-OES was conducted using a Spectro-Ciros-Vision Optical Emission Spectrometer.

Figure 5. (a) comparison of the amount of S. aureus (CFU/ml) in BHI surrounding, or attached to polyamide 12 or antibacterial polyamide 12 (containing 1.0% B65003), (b) comparison of amount of P. aeruginosa (CFU/ml) in BHI surrounding, or attached to polyamide 12 or antibacterial polyamide 12 (containing 1.0% B65003), (c) comparison of the amount of S. aureus (CFU/ml) in PBS containing 1 mM reduced glutathione surrounding, or attached to polyamide 12 or antibacterial polyamide 12 (containing 1.0% B65003).

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Bacterial strains and growth conditions. Bacterial strains used were clinical isolates of Staphylococcus aureus (S235) and Pseudomonas aeruginosa (SOM1) from the Charles Cliford Dental Hospital, Sheield24. hese were maintained on Brain Heart Infusion (BHI) agar (Sigma-Aldrich) plates at 37 °C. Liquid cultures were in BHI broth (Oxoid). Species of bacteria come in a variety of “strains”. We chose to use “clinical isolates” (strains taken from infected patients) as opposed to strains sourced from a standard strain collection, as clinical isolates are more representative of the types of bacteria that are encountered in the real environment. BHI is a widely used medium derived from animal products it has all the nutrients that the bacteria need. As both P. aeruginosa and S. aureus are capable of living in a human host, we grew them at body temperature, 37 °C.

Incubation of parts with bacteria. Overnight cultures were diluted to OD600 = 0.01 (this is a proxy meas-urement for the number of bacteria in a liquid a – typical maximum is 10) in either PBS or BHI. his solution (5 ml) was then added to glass universals containing autoclaved test parts and incubated with agitation (so all of the test part was equally exposed) at 37 °C for 24 hours.

Release of bioボlms┻ Parts were washed twice in PBS to release poorly attached bacteria before being vor-texed for 30 s in 2 ml PBS to release the well attached bioilm.

Quantiボcation of colony forming units┻ Bacterial suspensions were serially ten-fold diluted 10:90 µl in PBS in 96-well microtitre plates. Each well in the plate then contained a number of bacteria that could be related back to the parent concentration. Subsequently, 20 µl of each dilution was spotted onto BHI agar, such that at suiciently high dilutions each individual bacterium could form a colony distinguishable from its neighbours, before overnight incubation and colony counting. Once bacteria had been counted at the dilution where the highest density of colonies could be distinguished, we were then able to calculate the number of bacteria in the parent culture using Eq. 1.

= × ×CFU/ml Count 10 50 (1)dilution

he term Colony Forming Units (CFU) is used, as with this method we determine the number of colonies, not individual bacteria and in some cases more than one bacterium may be aggregated together in a single CFU.

Figure 6. Comparison of metabolic activity between ibroblast monolayers containing parts, a disc made of polyamide 12 or one made of antibacterial polyamide 12 (containing 1.0% B65003). A Kruskal-Wallis test indicates no diference between groups.

Figure 7. Dimensions of a type I tensile test specimen according to ASTM D638.

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1 0SCIENTIFIC REPORTS | (2020) 10:892 | https┺【【doi┻org【のね┻のねばふ【sぱのひぶふ┽ねはね┽ひぴびふび┽ぱ


Eukaryotic cell culture conditions. To obtain human dermal ibroblasts, human skin was obtained anon-ymously for medical research, with written informed consent, from individuals undergoing elective surgery for breast reduction or abdominoplasty. All methods were carried out in accordance with relevant guidelines and regulations and all experimental protocols were approved by the NHS Research Ethics Committee (REC), refer-ence: 15/YH/017.

To isolate primary ibroblasts, dermis from donated skin was minced, then digested by 0.05% collagenase A in Dulbecco’s Modiied Eagle’s Medium (DMEM) overnight at 37 °C with 5% CO2. he cell suspension was cen-trifuged at 400 g and the pellet resuspended in medium (DMEM supplemented with 10% fetal calf serum (FCS), 0.25 mg.mL−1 glutamine, 0.625 µg.mL−1 amphotericin B, 100 I.U.mL−1 penicillin, and 100 µg.mL−1 streptomy-cin). Cells were cultured in ibroblast medium in T25 lasks and incubated at 37 °C with 5% CO2. Medium was changed every 2 days, and cells were passaged as needed.

Quantiボcation of metabolic activity in eukaryotic cells┻ Fibroblasts were seeded at 50,000 cells/ml in 12 well plates (1 ml/well) and cultured for 24 hours before parts were added. Ater a further 24 hours parts were removed and growth media was replaced with the same containing 10% Presto Blue (Invitrogen). his was allowed to develop for 40 minutes before readout on a Tecan Ininite 200 plate reader in luorescence mode with excitation at 535 nm and emission at 590 nm. he luorescence of the Presto Blue is proportional to the metabolic activity of the cells.

Data availabilityhe datasets generated during and/or analysed during the current study are available from the corresponding authors on reasonable request.

Received: 29 August 2019; Accepted: 21 December 2019;

Published: xx xx xxxx

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AcknowledgementsWe are indebted to Kurt Bonser, Wendy Birtwistle and Jason Heath for technical advice and support. his work was funded by EPSRC grant EP/R036748/1 - ‘When the drugs don’t work. Manufacturing our pathogen defenses’. James R. Wingham is supported by an EPSRC DTP PhD studentship.

Author contributionsAll authors planned experiments, analysed data and contributed to manuscript preparation. R.D. Turner, J.R. Wingham and T.E. Paterson carried out experiments.

Competing interestshe authors declare no competing interests.

Additional informationCorrespondence and requests for materials should be addressed to J.S. or C.M.

Reprints and permissions information is available at www.nature.com/reprints.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional ailiations.

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or

format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre-ative Commons license, and indicate if changes were made. he images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not per-mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. © he Author(s) 2020

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Use of silver-based additives for the development of antibacterial functionality in Laser Sintered polyamide 12 partsResults and DiscussionProcessability of material. Mechanical properties of parts. Part structure and composition. Composite parts release silver. Composite parts have an antibacterial effect against gram positive Staphylococcus aureus and gram negative Pseudomonas aeru ...Antibacterial effects do not require contact between parts and bacteria. Antibacterial effects are diminished in rich media. Composite parts do not have a cytotoxic effect.

ConclusionsMethodsMaterials. Laser sintering. Mechanical properties. Scanning electron microscopy. Energy-dispersive x-ray spectroscopy. Micro-CT. Inductively coupled plasma – optical emission spectroscopy. Bacterial strains and growth conditions. Incubation of parts with bacteria. Release of biofilms. Quantification of colony forming units. Eukaryotic cell culture conditions. Quantification of metabolic activity in eukaryotic cells.

AcknowledgementsFigure 1 Engineering properties of parts (a) Photograph of a selection of parts made from PA2200 (left) alongside the 1% B65003 composite material (right).Figure 2 Images of parts – (a,b) SEM images of base material (sintered PA2200), (c,d) composite material.Figure 3 SEM and EDX map of composite PA2200 + B65003 surface.Figure 4 Comparison of amount of S.Figure 5 (a) comparison of the amount of S.Figure 6 Comparison of metabolic activity between fibroblast monolayers containing parts, a disc made of polyamide 12 or one made of antibacterial polyamide 12 (containing 1.Figure 7 Dimensions of a type I tensile test specimen according to ASTM D638.Table 1 Composition of the silver phosphate glass additive (B65003) and the composite part (PA2200 + 1.Table 2 ICP-OES measurements of silver concentration.

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Use of silver-based additives for the development of ...eprints.whiterose.ac.uk/156372/1/Antibacterial Paper.pdfWhatever the mechanism of antimicrobial action, our concern as engineers